DaXi—high-resolution, large imaging volume and multi-view single-objective light-sheet microscopy

The promise of single-objective light-sheet microscopy is to combine the convenience of standard single-objective microscopes with the speed, coverage, resolution and gentleness of light-sheet microscopes. We present DaXi, a single-objective light-sheet microscope design based on oblique plane illumination that achieves: (1) a wider field of view and high-resolution imaging via a custom remote focusing objective; (2) fast volumetric imaging over larger volumes without compromising image quality or necessitating tiled acquisition; (3) fuller image coverage for large samples via multi-view imaging and (4) higher throughput multi-well imaging via remote coverslip placement. Our instrument achieves a resolution of 450 nm laterally and 2 μm axially over an imaging volume of 3,000 × 800 × 300 μm. We demonstrate the speed, field of view, resolution and versatility of our instrument by imaging various systems, including Drosophila egg chamber development, zebrafish whole-brain activity and zebrafish embryonic development – up to nine embryos at a time.

O2. Move O2 and O3 together axially until at one point the magnification is uniform. The pupil plane of O2 is then conjugate to that of O1.
18. Translate the stages to move O2 and O3 together axially until at one point the magnification is uniform.
19. Place a sample with beads imbedded in 0.5% agarose on the sample stage. Place the emission filter, switch on the laser and observe the fluorescence image of the beads. The defocused image of the beads should look circular, otherwise slightly adjust O2 laterally to have a circular image of the beads. Adjust O3 slight to bring back the field of view to the centre of the camera. One can move O3 axially to inspect the images of the beads along different z. The image quality should be constant across at least 500 μm, otherwise the alignment is subject to further trouble shooting and improvement.
Dual view component alignment. 20. Adjust the voltages sent to the switching mirrors so that the light is reflect to the centre of the M6 and also passes through the irises from M6 to O1 thought the centre.
21. Place the grid back to the sample stage and measure the magnification with O3 at 200 μm away and closer to O2. Translate the stage to move M6 so that the magnification is uniform when translating O3 axially.

Repeat step 20.
Oblique light sheet setup and alignment.
23. Place O3 and the components downstream at 45° to O2. Place a sample with beads imbedded in 0.5% agarose on the sample stage. Adjust O3 so that the fluorescence image of the beads is capture by the camera in the centre of the field of view.
24. Set up CL1 and CL2 so that the laser light is expanded along the direction horizontal to the optical table.
25. Set up CL3 so that the beam is focused on the 2-axes galvo.
26. Place the slit at approximately the focal plane of CL3.
27. Place L3 so that the light becomes collimated again along the vertical direction.
28. Place a sample with beads imbedded in 0.5% agarose on the sample stage. Adjust the sample of the y-axes galvo to adjust the incident angle of the light sheet to 45° within the xy plane. Adjust O3 slightly to refocus if necessary. When the light sheet is at the correct angle, all beads in the image will appear in focus.

Supplementary Note 3. AMS-AGY v2.0 Objective -Full technical details and context.
Light-sheet fluorescence microscopy (LSFM) or selective plane illumination microscopy (SPIM) is a powerful technique for biological imaging. Fast, gentle, and with good sectioning, the idea has seen much attention with innovations like the DiSPIM 1 and Lattice 2 , and commercial instruments like the Zeiss Lightsheet 7 and the Leica DLS. However, traditional light-sheet designs require two or more orthogonal lenses for illumination and collection, resulting in an awkward interface between the biology and the optics, and a major drawback for many users and applications.
The Oblique Plane Microscope (OPM) 3 invention of 2008 restored the traditional coverslip boundary by passing light-sheet excitation and emission through a single primary objective, and then using a tilted remote refocus (RR) in the downstream optics to image the equally tilted plane of illumination (the object plane). OPM showed that light-sheet microscopy could be done with a standard microscope and sample interface, but seemingly exchanged this convenience for heavy losses in resolution and optical efficiency.
The problem with OPM was that the tilted portion of the remote refocus would lose a significant fraction of the emission light, simply because the numerical aperture of the final objective was too low. An ideal objective for this location would collect all the emission light, even with the additional tilt imposed by the OPM architecture, i.e. a full hemisphere of collection (a seemingly impossible requirement when objective half angles are typically limited to 70 deg).
However, in 2018 the Epi-illumination SPIM microscope (eSPIM) 4 showed that the major optical losses in OPM style systems could in fact be avoided. By using a water objective and coverslip assembly as the final objective, the numerical aperture could now equal the refractive index of air (1.0) i.e. the 'immersion medium' of the opposing objective in the remote refocus. This is the crucial insight: to make the elusive 'hemisphere' collection objective you simply need a numerical aperture that is greater than (or equal to) the index of the medium in which it operates. So for example, an NA 1.0 water immersion objective has a modest 49 deg collection cone in water (a reasonable lens to manufacture), but in air this transforms to 90 deg. So by imaging at the coverslip boundary, a water lens can indeed collect a solid angle of 2pi from an air medium. There are however some additional considerations that complicate the eSPIM approach. The tertiary objective assembly is corrected for a water/coverslip/water medium (not water/ coverslip/air). It can perform well if operated exactly at the surface of the coverslip, but deviations in alignment that push the image into the coverslip (or out into the air) will produce strong aberrations, so it can be challenging to align and keep stable (and hydrated). In addition, the bulkiness of the coverslip-water assembly requires longer working distance objectives in the remote refocus to avoid mechanical collision from the tilt. In practice this limits the choice of optics (and tilt range) and can force a reduced numerical aperture on the air objective.
Inspired by OPM and eSPIM, the AMS-AGY v1.0 objective (aka Snouty) 5,6 was developed to eliminate the previous trade-offs and compress the opto-mechanical difficulty into a single dedicated component (Supp. Fig. 5B). The NA 1.0 objective features a monolithic glass tip with zero working distance; this tip is optically equivalent to an oil/coverslip/air interface but alignment-free and mechanically stable. The tip also features an anti-reflection coating to maximize collection from the full hemisphere of rays (as noted previously NA 1.0 in an air collects from the full 90 deg half-angle). The zero working distance is another critical feature; the large refractive index mismatch at the glass-air interface produces strong spherical aberrations that vanish only at this boundary. The high refractive index of the glass tip compresses the collection half-angle so the tip can be shaved off (Supp. Fig. 5) to allow a range of tilt angles from 0-45deg. This excellent mechanical clearance allows image collection as close as 100um from a planar boundary. In practice this means the AMS-AGY objective can be paired with objectives with the highest numerical apertures and therefore the maximum collection efficiency. Infinity and color corrected, this component enables an extensive suite of design options as detailed in the High NA single-objective light-sheet (SOLS) article of 2019.
The Snouty v1.0 objective enabled 'bolt-on' SOLS designs with uncompromised numerical aperture, and is the ideal microscope for many light-sheet applications. However, the design of the v1.0 lens was constrained by mechanical and economic considerations, ultimately limiting the field of view (FOV) to 150um diffraction-limited (250um to the shaved edge, Fig.  5B). The economic argument is obvious: manufacturing difficulty and cost typically increase with field of view, and a costly lens would increase the prototyping risk and lower uptake of the technology. The mechanical limitations are more subtle; for the highest numerical aperture SOLS designs, the air objective that opposes the Snouty lens in the remote refocus can have working distances as short as 200um. So as the Snouty lens is tilted, the field of view (housed in glass) moves towards a collision with the opposing lens i.e. the Snouty FOV competes directly with the working distance of the paired objective in the remote refocus.
To overcome the limits on field of view the Snouty v2.0 prototype (aka KingSnout) was developed with a 3x boost on FOV compared to the v1.0 lens i.e. 450um diffraction limited and 450um to the shaved edge (Supp. Fig. 6&7). The large field was partly achieved by increasing the budget for design and manufacture (it is a larger and more complex lens) but also by eliminating the margin between the diffraction limited FOV and the shaved edge. This enables the lens to be used 'off-axis' in the tightest of spaces without losing optical quality. Snouty v2.0 is also ground at a more aggressive angle (55 deg vs 45 deg, see Supp. Fig. 6A) which combined with the reduced margins gives the maximum clearance for tilting in the remote refocus. It is a strict upgrade to the v1.0 objective and compatible with all SOLS designs. The discussion of lens specifications brings to the forefront some important considerations that should be emphasized.
The AMS-AGY objectives are exactly as specified: NA 1.0 with fields of 150um (v1.0) and 450um (v2.0), where NA 1.0 actually means the lens will image stigmatically at NA 1.0, not that it merely collects at this NA. How these specifications translate into the object space and the resulting volumetric imaging performance is subtle and beyond the scope of this section. However it should be noted that these objectives can be used beyond the specified fields, by up to a factor of approximately 1.7x, but not at NA 1.0. So a less genuine, but perhaps more typical specification would be NA 1.0 with fields of 250 um (v1.0) and 750 um (v2.0) and is something to bear in mind when considering a SOLS design. Water dispenser for water immersion lens. For long-term imaging, we built a water dispenser to automatically supply immersion water between the primary objective and the sample. A micropump (part of a LeicaWater Immersion Micro Dispenser) pumps water through a microcapillary tip (Eppendorf Microloader) to supply the immersion water to the primary objective. A custom-designed objective cap is 3D-printed using an elastic resin (Elastic 50A, Formlabs). The cap on the objective serves as water reservoir in case of excessive water supply from the pump. The microcapillary tip is glued on the cap for support. The micropump is controlled via serial command to supply water at desired time intervals during imaging.  1. With galvo scanning, the imaging quality varies cross the scan range because the illuminated plane is moving with respect to the optical axis. The more this plane moves away from the optical axis, the faster the image quality degrades. With stage scanning, this issue is avoided as the illuminated plane is static with respect to the optical axis, or within a few μm in case of LS3.
Supplementary Figure 8. Simulation of the temporal resolution of the microscope comparing tiled galvo scanning versus LS 3 scanning. (a) The time required to acquire a 3D dataset for LS 3 scanning as a function of the scan range is: Tvolume = nframes * tacquistion + tflyback + tadditional, where Tvolume is the time per volume, nframes is the number of frames (2D images) within the 3D volume, tacquistion is the acquisition time per frame, tflyback is the time for the stage to go back to the initial position (or next position in case of multiple position imaging) after each scan. Moreover, tadditional is the additional time needed for stage movement including the time required for software communication between computer and stage, for the stage to settle mechanically, and for the stage to accelerate and deaccelerate. Finally, nframes is equal to the scan range divided by the scan step and tacquistion is the exposure time plus the readout time of the camera. (b) Imaging time comparison of LS 3 scanning versus tiled galvo scanning as a function of the length of the volume. Imaging time for tiled galvo stage takes into consideration of both the time required to acquire each tile (corresponds to a 300 μm galvo scan), and the time needed to move the stage between tiles and after the last tile. When the volume length to be imaged is 300 μm (or less), no tiling is needed, and galvo scan is faster. However, when the volume to be imaged is larger than 300 μm along the main axis, tiled scan is required and leads to slower imaging than LS 3 scanning. This is mostly due to the multiple movements of the stage between consecutive tiles and after the last tile to go back to the starting position. Note LS 3 is as fast as continuous stage scanning but does not suffer from its drawbacks such as motion blur, photodamage or bleaching (when used in conjunction with light-sheet strobing to reduce motion-blur). Other key parameters for the simulation are: the scan step is set to 1.2 μm; The readout time per frame is 5 ms (for a region of interest of 1024 * 2048 pixels on the camera); The maximum scan speed of the stage is 7 mm/s; tadditional is set to 500 ms, a reasonable number considering that the acceleration and deacceleration time is ~ 100 ms, and the settling time is also on the order of 100 ms. show the simulated PSF and pupil function for the optical system presented in this paper. When the angle between the two objectives is not 0_, the effective pupil function of the imaging system shows a compression of the light towards one dimension. The resulting PSF is therefore not straight along the z axis, but has an angle to it, which is 10° for (b) and 20° for (d). The PSFs are fitted by a 1D Gaussian Function along the three principal axes to obtain the FWHMs, given at the bottom of the images. The tilted PSFs (b and d) are first rotated to have their principal axes along the x-, y-and z-axes and then fitted with 1D Gaussian Function to get the FWHMs. The FWHMs along y are comparable with O2 and O3 either along a straight line or tilted, suggesting that the effective NA is the same as the secondary objective. The FWHMs along the two other directions are slightly wider when the two objectives are tilted due to the asymmetry of the pupil function. Note that the FWHMs are adjusted by 1.33 to reflex the magnification from the primary objective (water) to the secondary objective (air). 2. The modified etendue is defined as the width of the field of view (y-axis) * the depth of the field of view (z-axis) * detection NA (x-axis) * detection NA (y-axis).

Supplementary
The modified etendue here gives the theoretical upper bound of the optical throughput of those methods independently of particular implementations such as choice of camera.
Practical comparison among those methods would depend on the particular implementations of each method, for example an estimation of the effective spatial resolution across the whole imaging volume, which is not available for many of the methods.
In our current system, the full field of view is sampled at 440 nm due to limited number of pixel of the camera (2048*2048). Using a camera with smaller pixel size and more pixels would increase the practical throughput of our system.